Enzymatic synthesis of deoxyribonucleosides

ABSTRACT

The present invention relates to a method for the in vitro synthesis of deoxyribonucleosides and enzymes suitable for this method.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 10/049,750filed Dec. 9, 2002, incorporated herein by reference in its entirety,which is a §371 of PCT/EP00/08088 filed Aug. 18, 2000, which claimspriority from European Patent Application No. 99 116 425.2 filed Aug.20, 1999.

DESCRIPTION

The present invention relates to a method for the in vitro enzymaticsynthesis of deoxyribonucleosides and enzymes suitable for this method.

Natural deoxyribonucleosides (deoxyadenosine, dA; deoxyguanosine, dG;deoxycytidine, dC and thymidine, dT) are building blocks of DNA. TheN-glycosidic bond between nucleobase and sugar involves the N₁ of apyrimidine or the N₉ of a purine ring and the C₁, of deoxyribose.

In the living cells the four deoxyribonucleosides (dN) result from the“salvage pathway” of nucleotide metabolism. A group of enzymes isinvolved in cellular catabolism of deoxyribonucleosides. Besidesdeoxyriboaldolase (EC 4.1.2.4) and deoxyribomutase (EC 5.4.2.7), thisgroup also includes thymidine phosphorylase (EC 2.4.2.4) and purinenucleoside phosphorylase (EC 2.4.2.1). These four enzymes are induced bythe addition of deoxyribonucleosides to the growth medium. The genescoding for these enzymes have been shown to map closely together on thebacterial chromosome (Hammer-Jesperson and Munch-Peterson, Eur. J.Biochem. 1 7 (1970), 397 and literature cited therein). In E. coli thegenes as described above are located on the deo operon which exhibits anunusual and complicated pattern of regulation (Valentin-Hansen et al.,EMBO J. 1 (1982), 317).

Using the enzymes of the deo operon for synthesis of deoxynucleosideswas described by C. F. Barbas III (Overproduction and Utilization ofEnzymes in Synthetic Organic Chemistry, Ph.D. Thesis (1989), Texas A&MUniversity). He applied phosphopentomutase and thymidine phosphorylasefor the synthesis of deoxynucleosides. Deoxyribose 5-phosphate wasprepared by chemical synthesis (Barbas III et al., J. Am. Chem. Soc. 112(1990), 2013-2014), which makes this compound expensive as startingmaterial and not suitable for large scale synthesis. He also madedeoxyriboaldolase available as a recombinant enzyme and investigated itssynthetic applicability but neither he nor C. H. Wong (MicrobialAldolases in Carbohydrate Synthesis: ACS Symp. Ser. No. 466: Enzymes inCarbohydrate Synthesis, Eds. M. D. Bednarski, E. S. Simon (1991), 23-27)were able to carry out a coupled one-pot synthesis employing all threeenzymes. It appears likely that some drawbacks exist which could not becircumvented. Among these drawbacks are insufficient chemicalequilibrium, instability of intermediates, such as deoxyribose1-phosphate and inactivation and inhibition effects of involvedcompounds on the enzymes.

Evidence of an advantageous equilibrium is given by S. Roy et al. (JACS108 (1986), 1675-78). For the aldolase reaction the equilibrium is onthe desired product side (deoxyribose 5-phosphate), for thephosphopentomutase it is on the wrong side (also deoxyribose5-phosphate) and for the purine nucleoside phosphorylase it is on thedesired synthesis product side. The authors suggest coupling of thethree enzyme reactions to obtain reasonable yields. Contrary to thesesuggestions they prepared deuterated deoxyguanosine and thymidine in atwo step procedure, that is deoxyribose 5-phosphate in a first step anddeoxynucleoside in a second step. Isolated yields of the second stepwere 11% and 5% for deoxyguanosine and thymidine, respectively. Theselow yields are also obtained in the preparation of arabinose-basednucleosides (Barbas III (1990), supra). These low yields indicateserious drawbacks for the use of the enzymes of the deo operon in asynthetic route which have to work in the reverse direction of theirbiological function, which is degradation of deoxynucleosides.

Thus, there does not exist any economical commercial method at presentfor the enzymatic in vitro synthesis of deoxyribonucleosides. Hitherto,for commercial purposes, deoxynucleosides are generated from fish spermby enzymatic cleavage of DNA. This method, however, involves severaldisadvantages, particularly regarding difficulties of obtaining thestarting material in sufficient quantity and quality.

Therefore, it was an object of the invention to provide a method, bymeans of which the drawbacks of the prior are eliminated at leastpartially and which allows efficient and economical synthesis ofdeoxyribonucleosides without any dependence on unreliable naturalsources.

Surprisingly, it was found that the drawbacks of previous enzymaticsynthesis routes can be avoided and deoxyribonucleosides can be obtainedin high yields of e.g. at least 80% based on the amount of startingmaterial.

In a first aspect, the present invention relates to a method for the invitro enzymatic synthesis of deoxyribonucleosides comprising reactingdeoxyribose 1-phosphate (dR1P) and a nucleobase, wherein adeoxyribonucleoside and inorganic phosphate are formed.

The reaction is catalyzed by an enzyme which is capable of transferringa deoxyribose moiety to a nucleobase, with a deoxyribonucleoside beingformed. Preferably, the reaction is catalyzed by a thymidinephosphorylase (TP, EC 2.4.2.4) or a purine nucleoside phosphorylase(PNP, EC 2.4.2.1). For the EC designation of these enzymes and otherenzymes mentioned below reference is made to the standard volume EnzymeNomenclature 1992, Ed. E. C. Webb, Academic Press, Inc.

These enzymes and other enzymes mentioned below are obtainable as nativeproteins from natural sources, i.e. any suitable organisms selected fromeukaryotes, prokaryotes and archaea including thermophilic organisms.Further, these enzymes are obtainable as recombinant proteins from anysuitable host cell which is transformed or transfected with a DNAencoding said enzyme. The host cell may be a eukaryotic cell, aprokaryotic cell or an archaea cell. Particular preferred sources ofnative or recombinant TP or PNP are prokaryotic organisms such as E.coli. Recombinant TP may be isolated from E. coli strain pHSP 282 (CNCMI-21 86) deposited on Apr. 23, 1999, which is a recombinant E. colistrain transformed with a plasmid containing the E. coli deoA (thymidinephosphorylase) insert. Recombinant PNP may be isolated from E. colistrain pHSP 283 (CNCM 1-2187) deposited on Apr. 23, 1999, which is arecombinant E. coli strain transformed with a plasmid containing the E.coli deoD (purine nucleoside phosphorylase) insert. The nucleotidesequence of the TP gene and the corresponding amino acid sequence areshown in SEQ ID NO.1 and 2. The nucleotide sequence of the PNP gene andthe corresponding amino acid sequence are shown in SEQ ID NO.15 and 16and 3 and 4.

The nucleobase, to which the deoxyribose unit is transferred, will beselected from any suitable nucleobase. For example, the nucleobase maybe a naturally occurring nucleobase such as thymine, uracil, adenine,guanine or hypoxanthine. It should be noted, however, that alsonon-naturally occurring analogs thereof are suitable as enzymesubstrates such as 2-thio-uracil, 6-aza-uracil, 5-carboxy-2-thiouracil,6-aza-thymine, 6-aza-2-thio-thymine and 2,6-diamino-purine.

Preferably the inorganic phosphate is removed from the reaction. Thisremoval is preferably effected by (i) conversion to inorganicpyrophosphate, (ii) precipitation/complexation and/or (iii) substratephosphorylation.

Conversion to inorganic pyrophosphate may be effected by a phosphatetransfer from a phosphorylated, preferably polyphosphorylated substratesuch as fructose diphosphate (FDP), wherein a phosphate group is cleavedfrom the phosphorylated substrate and reacts with the inorganicphosphate, with inorganic pyrophosphate (PPi) being formed. Thisphosphate transfer is preferably catalyzed by a PPi-dependentphosphorylase/kinase, e.g. by a PPi-dependent phosphofructokinase(PFK-PPi, EC 2.7.1.90), which catalyzes the reaction of fructosediphosphate (FDP) and inorganic phosphate to fructose 6-phosphate (F6P)and inorganic pyrophosphate. Preferred sources of PPi-dependentkinases/phosphorylases and genes coding therefor are fromPropionibacterium freudenreichii (shermanii) or from potato tubers.

Further, the inorganic phosphate may be removed from the reaction byprecipitation and/or complexation which may be effected by addingpolyvalent metal ions, such as calcium or ferric ions capable ofprecipitating phosphate or by adding a complex-forming compound capableof complexing phosphate. It should be noted that also a combination ofpyrophosphate formation and complexation/precipitation may be carriedout.

Furthermore, the removal of inorganic phosphate may be effected bysubstrate phosphorylation. Thereby the inorganic phosphate istransferred to a suitable substrate, with a phosphorylated substratebeing formed. The substrate is preferably selected from saccharides,e.g. disaccharides such as sucrose or maltose. When using disaccharidesas substrate, a monosaccharide and a phosphorylated monosaccharide areobtained. The phosphate transfer is catalyzed by a suitablephosphorylase/kinase such as sucrose phosphorylase (EC 2.4.1.7) ormaltose phosphorylase (EC 2.4.1.8). Preferred sources of these enzymesare Leuconostoc mesenteroides, Pseudomonas saccherophila (sucrosephosphorylase) and Lactobacillus brevis (maltose phosphorylase).

The phosphorylated substrate may be further reacted by additionalcoupled enzymatic reactions, e.g. into a galactoside (Ichikawa et al.,Tetrahedron Lett. 36 (1995), 8731-8732). Further, it should be notedthat phosphate removal by substrate phosphorylation may also be coupledwith other phosphate removal methods as described above.

Deoxyribose 1-phosphate (dR1P), the starting compound of the method ofthe invention, is a rather unstable compound, the isolation of which isdifficult. In a preferred embodiment of the present invention, d1RP isgenerated in situ from deoxyribose 5-phosphate (dR5P) which isrelatively stable at room temperature and neutral pH. This reaction iscatalyzed by a suitable enzyme, e.g. a deoxyribomutase (EC 5.4.2.7) or aphosphopentose mutase (PPM, EC 5.4.2.7) which may be obtained from anysuitable source as outlined above. The reaction is preferably carriedout in the presence of divalent metal cations, e.g. Mn²⁺ or Co²⁺ asactivators. Preferred sources of deoxyribomutase are enterobacteria.Particular preferred sources of native or recombinant PPM areprokaryotic organisms such as E. coli. Recombinant PPM may be isolatedfrom E. coli strain pHSP 275 (CNCM I-2188) deposited on Apr. 23, 1999,which is a recombinant E. coli strain transformed with a plasmidcontaining the E. coli deo B (phosphopentose mutase) insert. Thenucleotide sequence of the PPM gene and the corresponding amino acidsequence are shown in SEQ ID NO.17 and 18 and 5 and 6.

dR5P may be generated by a condensation of glyceraldehyde 3-phosphate(GAP) with acetaldehyde. This reaction is catalyzed by a suitableenzyme, preferably by a phosphopentose aldolase (PPA, EC 4.1.2.4). Thereaction exhibits an equilibrium constant favorable to the formation ofthe phosphorylated sugar(K_(eq)=[dR5P]/[acetaldehyde]×[GAP]=4.2×10³×M⁻¹). PPA forms an unstableSchiff base intermediate by interacting with the aldehyde. Particularpreferred sources of native or recombinant PPA are prokaryotic organismssuch as E. coli. Recombinant PPA may be isolated from E. coli strainpHSP 276 (CNCM I-2189) deposited on Apr. 23, 1999. This recombinant E.coli strain is transformed with a plasmid containing the deoC(phosphopentosealdolase) insert. The nucleotide sequence of the PPA geneand the corresponding amino acid sequence are shown in SEQ ID NO.19 and20 and 7 and 8.

GAP is a highly unstable compound and, thus, should be generated in situfrom suitable precursors which are preferably selected from fructose1,6-diphosphate (FDP), dihydroxyacetone (DHA) and/or glycerolphosphate(GP), with FDP being preferred.

FDP can be converted by an FDP aldolase (EC 4.1.2.13) selected from FDPaldolases I and FDP aldolases II to GAP and dihydroxyacetone phosphate(K_(eq)=[FDP]/[GAP]×[DHAP]=10⁴M⁻¹). The two families of FDP aldolasesgiving identical end products (GAP and DHAP) via two chemically distinctpathways may be used for this reaction. FDP aldolase I forms Schiff baseintermediates like PPA, and FDP aldolase II which uses metals (Zn²⁺)covalently bound to the active sites to generate the end products.FDP-aldolase I is characteristic to eukaryotes, although it is found invarious bacteria. FDP-aldolase II is more frequently encountered inprokaryotic organisms. If FDP-aldolase reacts with FDP in the presenceof acetaldehyde, the latter compound can interact with DHAP to yield anundesired condensation by-product named deoxyxylolose 1-phosphate(dX1P). Thus, the reaction is preferably conducted in a manner by whichthe generation of undesired side products is reduced or completelysuppressed.

Particular preferred sources of native or recombinant FDP aldolases areprokaryotic or eukaryotic organisms. For example, FDP aldolase may beisolated from rabbit muscle. Further, FDP aldolase may be obtained frombacteria such as E. coli. Recombinant FDP aldolase may be isolated fromrecombinant E. coli strain pHSP 284 (CNCM I-2190) which is transformedwith a plasmid containing the E. coli fba (fructose diphosphatealdolase) insert. The nucleotide sequence of the E. coli FDP aldolasegene and the corresponding amino acid sequence are shown in SEQ ID NO.9and 10.

On the other hand, GAP may be generated from DHAP and ATP, withdihydroxyacetone phosphate (DHAP) and ADP being formed and subsequentisomerization of DHAP to GAP in a reaction catalyzed by a glycerokinase(GK, EC 2.7.1.30) and a triose phosphate isomerase (TIM, EC 5.3.1.1).Suitable glycerokinases are obtainable from E. coli, suitable triosephosphate isomerases are obtainable from bovine or porcine muscle.

In a still further embodiment of the present invention GAP may begenerated from glycerol phosphate (GP) and O₂, with DHAP and H₂O₂ beingformed and subsequent isomerization of DHAP to GAP in a reactioncatalyzed by a glycerophosphate oxidase (GPO, EC 1.1.3.21) and a triosephosphate isomerase (TIM, EC 5.3.1.1). Suitable glycerophosphateoxidases are obtainable from Aerococcus viridans.

In an alternative embodiment of the present invention deoxyribose5-phosphate (dR5P) is generated by phosphorylation of deoxyribose.Preferably this reaction is carried out in the presence of a suitableenzyme, e.g. a deoxyribokinase (dRK, EC 2.7.1.5) which may be obtainedfrom prokaryotic organisms, particularly Salmonella typhi and in thepresence of ATP. The nucleotide sequence of the Salmonella dRK gene andthe corresponding amino acid sequence are shown in SEQ ID NO.11 and 12.

By the reaction as outlined above deoxyribonucleosides are obtainedwhich contain a nucleobase which is accepted by the enzymes TP and/orPNP. TP is specific for thymidine (T), uracil (U) and other relatedpyrimidine compounds. PNP uses adenine, guanine, hypoxanthine or otherpurine analogs as substrates.

The synthesis of deoxyribonucleosides which are not obtainable by directcondensation such as deoxycytosine (dC), thus, require an additionalenzymatic reaction, wherein a deoxyribonucleoside containing a firstnucleobase is reacted with a second nucleobase, with a secondribonucleoside containing the second nucleobase being formed. The secondnucleobase is preferably selected from cytosine and analogs thereof suchas 5-azacytosine. It should be noted, however, that also othernucleobases such as 6-methyl purine, 2-amino-6-methylmercaptopurine,6-dimethylaminopurine, 2,6-dichloropurine, 6-chloroguanine,6-chloropurine, 6-azathymine, 5-fluorouracil, ethyl-4-amino-5-imidazolecarboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamidemay be converted to the corresponding deoxyribonucleoside by thisnucleobase exchange reaction (Beaussire and Pochet, Nucleosides &Nucleotides 14 (1995), 805-808, Pochet et al. Bioorg. Med. Chem. Lett. 5(1995), 1679-1684, Pochet and Dugue, Nucleosides & Nucleotides 17(1998), 2003-2009, Pistotnik et al., Anal. Biochem. 271 (1999),192-199). This reaction is preferably catalyzed by an enzyme callednucleoside 2-deoxyribosyltransferase (NdT, EC 2.4.2.6) which transfersthe glycosyl moiety from a first deoxynucleoside to a second nucleobase,e.g. cytosine. A preferred source of native or recombinant NdT areprokaryotic organisms such as lactobacilli, particularly Lactobacillusleichmannii. Recombinant NdT may be isolated from recombinant E. colistrain pHSP 292 (CNCM I-2191) deposited on Apr. 23, 1999, which istransformed with a plasmid containing the L. leichmannii NdT (nucleoside2-deoxyribosyltransferase) insert. The nucleotide sequence of the NdTgene and the corresponding amino acid sequence are shown in SEQ ID NO.13and 14.

A further aspect of the present invention is a method for the in vitroenzymatic synthesis of deoxyribonucleosides comprising the steps of: (i)condensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde todeoxyribose 5-phosphate (dR5P), (ii) isomerizing deoxyribose 5-phosphateto deoxyribose 1-phosphate (dRIP) and (iii) reacting deoxyribose1-phosphate and a nucleobase, wherein a deoxyribonucleoside andinorganic phosphate are formed. Preferably, the reaction is carried outwithout isolating intermediate products and, more preferably, as aone-pot reaction. Further, the removal of the inorganic phosphate fromthe reaction is preferred.

As outlined above, the glyceraldehyde 3-phosphate may be generated fromFDP, DHA and/or GP. Preferably, FDP is used as a starting material.

In order to avoid the production of undesired by-products and the toxiceffects of acetaldehyde, the course of the reaction is preferablycontrolled by suitable means. Thus, preferably, the reaction is carriedout in a manner such that the acetaldehyde concentration in step (ii) iscomparatively low, e.g. less than 100 mM, particularly less than 50 mM,e.g. by adding the acetaldehyde in portions or continuously during thecourse of the reaction and/or by removing excess acetaldehyde. Further,it is preferred that before step (ii) excess starting materials and/orby-products, particularly fructose 1,6-diphosphate and/or deoxyxylulose1-phosphate (dX1P), are removed. This removal may be effected bychemical and/or enzymatic methods, e.g. precipitating FDP with ferricsalts or enzymatically degrading X1P via dihydroxyacetone phosphate.Alternatively or additionally the reaction conditions may be adjustedsuch that before step (ii) no substantial amounts, preferably less than10 mM, of starting materials and/or by-products, particularly fructose1,6-diphosphate and/or deoxyxylulose 1-phosphate, are present in thereaction mixture.

In still another embodiment, the present invention relates to a methodfor the in vitro enzymatic synthesis of deoxyribonucleosides comprisingthe steps of: (i) phosphorylating deoxyribose to deoxyribose5-phosphate, (ii) isomerizing deoxyribose 5-phosphate to deoxyribose1-phosphate and (iii) reacting deoxyribose 1-phosphate and nucleobase,wherein a deoxyribonucleoside and inorganic phosphate are formed.Preferably, these reactions are carried out with isolating intermediateproducts and, more preferably, as a one-pot reaction. To obtain a betteryield the removal of inorganic phosphate from step (iii) is preferred.

By the process as described above naturally occurringdeoxyribonucleosides such as dA, dG, dT, dU and dT but also analogsthereof containing non-naturally occurring nucleobases and/ornon-naturally occurring deoxyribose sugars such as2′-deoxy-3′-azido-deoxyribose or 2′-deoxy-4′-thio-deoxy-ribose may beproduced.

The deoxyribonucleosides obtained may be converted to further productsaccording to known methods. These further reaction steps may comprisethe synthesis of deoxyribonucleoside mono-, di- or triphosphates, ofH-phosphonates or phosphoramidites. Additionally or alternatively,labelling groups such as radioactive or chemical labelling groups may beintroduced into the deoxyribonucleosides.

Still a further aspect of the present invention is the use of anisolated nucleic acid molecule encoding a nucleoside 2-deoxyribosyltransferase (NdT, EC 2.4.2.6) for the preparation of an enzyme in an invitro enzymatic synthesis process, wherein a deoxyribonucleosidecontaining a first nucleobase is reacted with a second nucleobase, witha deoxyribonucleoside containing the second nucleobase being formed. Thesecond nucleobase is preferably selected from cytidine and analogsthereof, 2,6-dichloro-purine, 6-chloro-guanine, 6-chloro-purine,6-aza-thymine and 5-fluoro-uracil. The first nucleobase is preferablyselected from thymine, guanine, adenine or uracil. More preferably, thenucleic acid molecule encoding an NdT comprises (a) the nucleotidesequence shown in SEQ ID NO.13 or its complementary sequence, (b) anucleotide sequence corresponding to the sequence of (a) in the scope ofdegeneracy of the genetic code or (c) the nucleotide sequencehybridizing under stringent conditions to the sequence (a) and/or (b).Apart from the sequence of SEQ ID NO.13 the present invention alsocovers nucleotide sequences coding for the same polypeptide, i.e. theycorrespond to the sequence within the scope of degeneracy of the geneticcode, and nucleotide sequence hybridizing with one of theabove-mentioned sequences under stringent conditions. These nucleotidesequences are obtainable from SEQ ID NO.13 by recombinant DNA andmutagenesis techniques or from natural sources, e.g. from otherLactobacillus strains.

Stringent hybridization conditions in the sense of the present inventionare defined as those described by Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989),1.101-1.104. According to this, hybridization under stringent conditionsmeans that a positive hybridization signal is still observed afterwashing for one hour with 1×SSC buffer and 0.1% SDS at 55° C.,preferably at 62° C. and most preferred at 68° C., in particular, forone hour in 0.2×SSC buffer and 0.1% SDS at 55° C., preferably at 62° C.and most preferred at 68° C.

Moreover, the present invention also covers nucleotide sequences which,on nucleotide level, have an identity of at least 70%, particularlypreferred at least 80% and most preferred at least 90% to the nucleotidesequence shown in SEQ ID NO.13. Percent identity are determinedaccording to the following equation:

$I = {\frac{n}{L} \times 100}$

wherein I are percent identity, L is the length of the basic sequenceand n is the number of nucleotide or amino acid matching between theselected sequence and that of the basic sequence.

Still another subject matter of the present invention is a recombinantvector comprising at least one copy of the nucleic acid molecule asdefined above, operatively linked with an expression control sequence.The vector may be any prokaryotic or eukaryotic vector. Examples ofprokaryotic vectors are chromosomal vectors such as bacteriophages (e.g.bacteriophage Lambda) and extrachromosomal vectors such as plasmids(see, for example, Sambrook et al., supra, Chapter 1-4). The vector mayalso be a eukaryotic vector, e.g. a yeast vector or a vector suitablefor higher cells, e.g. a plasmaid vector, viral vector or plant vector.Suitable eukaryotic vectors are described, for example, by Sambrook etal., supra, Chapter 16. The invention moreover relates to a recombinantcell transformed with the nucleic acid or the recombinant vector asdescribed above. The cell may be any cell, e.g. a prokaryotic oreukaryotic cell. Prokaryotic cells, in particular, E. coli cells, areespecially preferred.

The invention refers to an isolated polypeptide having NdT activityencoded by the above-described nucleic acid and its use for thepreparation of deoxyribonucleosides. Preferably, the polypeptide has theamino acid sequence shown in SEQ ID NO.14 or an amino acid sequencewhich is at least 70%, particularly preferred at least 80% and mostpreferred at least 90% identical thereto, wherein the identity may bedetermined as described above.

Finally, the present invention also relates to the use of isolatednucleic acid molecules having thymidine phosphorylase (TP), purinenucleoside phosphorylase (PNP), phosphopentose mutase (PPM),phosphopentose aldolase (PPA), FDP aldolase and deoxyribokinase (dRK)activity for the preparation of an enzyme for a method for the in vitrosynthesis of deoxynucleosides. Preferably, these nucleic acids areselected (a) from a nucleotide sequence shown in SEQ ID NO.1, 3, 5, 7, 9or 11 or their complementary sequences, (b) a nucleotide sequencecorresponding to a sequence of (a) within the scope of degeneracy of thegenetic code or (c) a nucleotide sequence hybridizing under stringentconditions to a sequence (a) and/or (b).

Isolated polypeptides having TP, PNP, PPM, PPA, FDP aldolase or dRKactivity encoded by the above-described nucleic acids may be used forthe preparation of deoxyribonucleosides. Preferably, these polypeptideshave the amino acid sequence shown in SEQ ID NO.2, 4, 16, 6, 18, 8, 20,10 or 12 or an amino acid sequence which is at least 70%, particularlypreferred at least 80% and most preferred at least 90% identicalthereto, wherein the identity may be determined as described above.

An isolated nucleic acid molecule encoding a dRK may be used for thepreparation of an enzyme for an in vitro method for the enzymaticsynthesis of deoxyribonucleosides comprising the step of phosphorylatingdeoxyribose to deoxyribose 5-phosphate, wherein said nucleic acidmolecule comprises (a) the nucleotide sequence shown in SEQ ID NO. 11 orits complementary sequence, (b) a nucleotide sequence corresponding tothe sequence of (a) in the scope of the degeneracy of the genetic codeor (c) a nucleotide sequence hybridizing under stringent conditions tothe sequence of (a) and/or (b). Correspondingly, an isolated polypeptidehaving dRK activity is suitable for an in vitro method for the enzymaticsynthesis of deoxyribonucleosides as outlined above.

The E. coli strains pHSP 282 (CNCM I-2186), pHSP 283 (CNCM I-2187), pHSP275 (CNCM I-2188), pHSP 276 (CNCM 2189), pHSP 284 (CNCM I-2190) and pHSP292 (CNCM I-2191) were deposited according to the regulations of theBudapest Treaty on Apr. 23, 1999 at the Collection Nationale de Culturede Microorganismes, Institut Pasteur, 25, Rue de Docteur Roux, 75724Paris Cedex 15.

DESCRIPTION OF FIGURES

FIG. 1 shows the synthesis of dR5P according to Example 12.

FIG. 2 shows the synthesis of deoxyadenosine according to Example 12.

FIG. 3 shows the synthesis of deoxyadensine according to Example 13.

FIG. 4 shows the synthesis of dG-NH₂ according to Example 14.

EXAMPLE 1 Sources of Enzymes

L-glycerol 3-phosphate oxidase (1.1.3.21) from Aerococcus viridans,sucrose phosphorylase (2.4.1.7), fructose 6-phosphate kinase (2.7.1.90)from Propionibacterium freudenreichii, rabbit muscle aldolase (RAMA),formate dehydrogenase, glycerolphosphate dehydrogenase (GDH),triosephosphate isomerase (TIM), catalase, glycerol 3-phosphate oxidaseand maltose phosphorylase were obtained from commercial sources (RocheDiagnostics, Sigma) or as described in the literature.

FDP aldolase II (4.1.2.13), phosphopentose aldolase (PPA, EC 4.1.2.4),phosphopentose mutase (PPM, EC 5.4.2.7), thymidine phosphorylase (TP, EC2.4.2.4), purine nucleoside phosphorylase (PNP, EC 2.4.2.1), nucleoside2-deoxyribosyl transferase (NdT, EC 2.4.2.6) were obtained from E. colistrains deposited at CNCM (see above).

EXAMPLE 2 Protocol of the Synthesis of Deoxyadenosine

Reaction mixture A was prepared by adding acetaldehyde (finalconcentration 250 mM), FDP aldolase II (0.5 U/ml), PPA (2.5 U/ml) to 20ml of 100 mM fructose-1,6-diphosphate (FDP), pH 7.6 and incubatingovernight at 4° C.

Mixture B was prepared by adding MnCl₂ (final concentration 0.6 mM),glucose 1,6-diphosphate (15 μM), PPM (1.5 U/ml), PNP (0.4 U/ml), SP (1.5U/ml) pentosephosphate aldolase, PPA (2 U/ml) and FDP aldolase II (0.5U/ml) to 10 ml 0.9 M sucrose, pH 7.6, at room temperature.

2 ml of A were added over B at a temperature of 20° C. After 1 hour 2.5ml A were added. After another hour 3.0 ml A were added. After another1.5 h 3.5 ml A were added. After another 1.5 h 4 ml A were added andafter another 1-1.5 h 5 ml A were added and left to stand overnight.

At each time of addition of A the amounts of FDP, dR5P, dX1P and dA inthe reaction mixture were determined and the yield was calculated. Theconcentration of acetaldehyde was kept between 20-30 mM. The results areshown in Table 1:

TABLE 1 Time Volume Concentrations (mM) Yield (mmol) (h) (ml) dR5P dAdX1P dA 0 12 4 0 1.2 0 1 12 3.4 3.2 1 0.04 2 14.5 7.9 8.0 2.6 0.12 3.517.5 13 16.2 4.3 0.28 5 21 11.7 21.7 0.46 6 25 23.7 0.59 22 30 11 40.413.2 1.21 30 30 50.3 1.51 54 30 8.9 60.6 1.82

The starting amount of FDP was 1.92 mmol. The amount after completion ofreaction was 0.150 mmol. Thus, 1.77 mmol were consumed, theoreticallycorresponding to 3.54 mmol equivalents dA. The amount of dA formed was1.82 mmol, leading to a yield of 51.4% based on the amount of FDP.

EXAMPLE 3 Removal of Excess FDP by Means of FeCl₃

1.4 g (2.55 mmol) trisodium-fructose-1,6-disphosphate-octahydrate and430 μl (335 mg, 7.6 mmol) acetaldehyde were dissolved in 15 ml of waterat 4° C. A pH of 7.9 was adjusted by means of sodium hydroxide solution.150 U pentosephosphate aldolase (PPA) were added, and cold water (4° C.)was added to give 20 ml. After addition of 50 U E. coli aldolase II themixture was stored at 4° C. After 2 h another 75 U PPA and 50 μlacetaldehyde (390 mg, 8.9 mmol) were added. After 20 h 500 Utriosephosphate isomerase (TIM) were added. After 120 h the solutioncontained about 68 mM FDP, about 12 mM dX1P and about 45 mM dR5P. Thereaction was stopped by adding 900 μl of a 2 M solution of iron(III)chloride in 0.01 M hydrochloric acid. The precipitate was centrifugedand washed, the resulting solution contained about 4 mM dX1P, about 9 mMFDP and about 25 mM dR5P.

EXAMPLE 4 Removal of Excess FDP and dX1P by Degradation Via DHAP

576 mg (1.05 mmol) trisodium-fructose-1,6-disphosphate-octahydrate weredissolved in 8 ml water, and the pH was adjusted at 8.1 by means ofsodium hydroxide solution. 75 U PPA and 27 U rabbit muscle aldolase(RAMA) were added, and water was added to give 10 ml. 570 μl (440 mg, 10mmol) acetaldehyde were added. The reaction was stored at 4° C. After100 h the solution contained about 110 mM dX1P, about 5 mM FDP and about85 mM dR5P (about 870 μmol). The reaction was stopped by addinghydrochloric acid until a pH of 2 was reached. After adding sodiumhydroxide solution to give a pH of 5.5 the solution was stored.

For removing dX1P the acetaldehyde was evaporated and the solution wasdiluted with water to reach 30 ml. It was mixed with 3 ml 2.65 M sodiumformate solution (8 mmol), and sodium hydroxide solution was added untila pH of 7.4 was reached. 23 U formate dehydrogenase (FDH), 6 mg NADH, 16U RAMA and 20 U glycerolphosphate dehydrogenase (GDH) were added.

After 24 h at room temperature the concentrations of dX1P and FDP arebelow 3 mM, the loss of dR5P is less than 10%.

EXAMPLE 5 Preparation of dR5P Via G3P

1.1 g (2.0 mmol) trisodium-fructose-1,6-disphosphate-octahydrate weredissolved in 8 ml water. 1.58 mol of a 2.65 M sodium formate solution(4.2 mmol) and 14.2 mg NADH were added. A pH of 7.0 was adjusted bymeans of NaOH. After addition of 36 U RAMA, 50 U triosephosphateisomerase (TIM), 34 U GDH and 35 U FDH water was added to give 12 ml.

After incubation of 40 h at room temperature the FDP content was below 3mM. The enzymes were denatured by acidification with hydrochloric acidto reach a pH of 2. Subsequently, the pH of the solution was adjusted at4 and the solids were centrifuged and filtered off, respectively.Through dilution during purification a total volume of 25 ml was reachedwhich contained about 160 mM of glycerol-3-phosphate (G3P).

4 ml of this solution (about 640 μmol G3P) were adjusted at a pH of 7.8by means of sodium hydroxide solution. 7.8 kU catalase, 500 U TIM and 13U glycerol 3-phosphate oxidase are added. The mixture was stirred veryslowly in an open flask. After 30 min 18 U PPA were added. Acetaldehydewas added in portions of 30 μl (23.5 mg, 530 μmol) after 30, 60, 120,180 and 240 min. After 24 h another 15 U PPA, 2.5 kU TIM and 100 μl (78mg, 1.8 mmol) acetaldehyde were added. After 30 h the batch is sealedafter addition of another 100 μl acetaldehyde. After a total of 45 h aconcentration of about 60 mM dR5P was achieved and the reaction iscompleted. For preparing 2′-deoxyadenosine (e.g. Example 7) excessacetaldehyde must be distilled off.

EXAMPLE 6 Preparation of a dR5P Solution Containing Small Amounts ofdX1P or FDP

A solution of 60 mmol/l FDP and 120 mmol/l acetaldehyde having pH 7.4was kept at a temperature of 15° C. 5 ml thereof were mixed with 4 Ualdolase II, 2 U TIM and 40 U PPA and kept at 15° C. After 4, 8.5, 16.5and 24 h 12 U PPA and 100 μl of a 34 vol.-% solution of acetaldehyde inwater (26.4 mg, 600 μmol) were added each. After 40 h the solution wasallowed to reach room temperature. After 90 h the reaction solution hadreached concentrations of about 3 mM FDP, about 4 mM dX1P and at least70 mM dR5P. For stopping the reaction and removing acetaldehyde about20% of the volume were distilled off.

EXAMPLE 7 Preparation of Deoxyadenosine (dA) from dR5P by Means ofBarium Acetate

dR5P was used in the form of a solution prepared according to Examples3-6. For instance, 10 ml of a solution of Example 6 diluted to have 70mM dR5P (700 μmol dR5P) were mixed with 40 mg (300 μmol) adenine, 41 μg(50 nmol) tetracyclohexylammonium-glucose-1,6-disphosphate, 396 μg (2μmol) manganese-II-acetate-tetrahydrate, 10 U pentosephosphate mutase(PPM) and 30 U purine-nucleoside phosphorylase (PNP). After 3 h another27 mg (200 μmol) adenine and 26 mg (100 μmol) barium acetate were added.

A further amount of 26 mg barium acetate was added after 4 h, one of 40mg adenine after 7 h. After 10 h the reaction was completed. Thesolution had a concentration of 45 mM dA.

EXAMPLE 8 Preparation of Deoxyadenosine (dA) from dR5P by Means ofSucrose Phosphorylase

10 ml of a solution of Example 6 diluted to 55 mM dR5P (550 μmol dR5P)were mixed with 81 mg (600 μmol) adenine, 41 μq (50 nmol)tetracyclohexylammonium-glucose-1,6-disphosphate, 396 μg (2 μmol)manganese-II-acetate-tetrahydrate, 10 U pentosephosphate mutase (PPM) 15U purine nucleoside phosphorylase (PNP), 25 U sucrose phosphorylase and340 mg (1 mmol) cane sugar.

After 3 h at room temperature the reaction was completed. The solutionhad a concentration of about 50 mM dA.

EXAMPLE 9 Preparation of Deoxyadenosine (dA) from dR5P by Means ofMaltose Phosphorylase

10 ml of a solution of dR5P diluted to 55 mM were mixed at pH 7.0 with81 mg (600 μmol) adenine, 41 μg (50 nmoles) glucose 1,6-diphosphate, 396μg (2 μmoles) manganese II-acetate tetrahydrate, 5 units pentosephosphate mutase (PPM), 10 units purine nucleoside phosphorylase, (PNP),20 units maltose phosphorylase and 1080 mg (3 mmoles) maltose.

After 12 h at room temperature the reaction was completed. The solutionhad a concentration of 49 mM dA.

EXAMPLE 10 Preparation of Deoxycytosine (dC) from dR5P by Means ofSucrose Phosphorylase

20 ml of a solution of dR5P diluted to 70 mM were mixed at pH 7.0 with5.4 mg adenine (0.04 mmoles), 155 mg cytosine (1.4 mmoles), 82 μg (100nmoles) glucose 1,6-diphosphate, 792 μg (4 μmoles) manganeseII-acetate-tetrahydrate, 20 units PPM, 30 units PNP, 50 units2-deoxyribosyl transferase (NdT), 50 units sucrose phosphorylase and2.05 g (6 mmoles) sucrose.

After 18 h at 30° C. the solution had a concentration of 62 mM dC.

EXAMPLE 11 Preparation of Deoxyguanosine (dG) from dR5P by Means ofSucrose Phosphorylase

20 ml of a solution of dR5P diluted to 70 mM were mixed at pH 7.0 with91 mg guanine (0.6 mmoles), 82 μg (100 nmoles) glucose 1,6-diphosphate,792 μg (4 μmoles) manganese II-acetate-tetrahydrate, 20 units PPM, 10units PNP, 20 units sucrose phosphorylase and 2.05 g (6 mmoles) sucrose.

After 18 h at 37° C. the dG formed corresponds to 0.5 mmoles.

EXAMPLE 12 Two Step Procedure of dA Synthesis

In the first step dR5P was prepared by adding FDP-Aldolase II (AldII)from E. coli, pentosephosphate aldolase (PPA) from E. coli andtriosephosphate isomerase (TIM) from E. coli tofructose-1,6-bisphosphate (FDP) and acetaldehyde (AcAld) essentiallyaccording to Ex. 6. FDP trisodium salt was mixed in a finalconcentration of 75 mM with AcAld (100 mM final concentration). The pHwas adjusted to 7.4 by addition of sodium hydroxide. The reaction wasstarted by adding PPA (12 kU/l), Ald II (0.3 kU/l) and TIM (2.5 kU/l).At 4 h 117 mM AcAld, at 7 h 117 mm AcAld, PPA 6 kU/l, TIM 2.5 kU/l andat 12 h 117 mM AcAld were added. The reaction was run at 21° C.Conversion was monitored by enzymatical assay using step by stepglycerol-3-phosphate dehydrogenase (GDH), rabbit muscle aldolase (RAMA),trisosephosphate isomerase (TIM), pentosephosphate aldolase (PPA) in thepresence of NADH (0.26 mM in 300 mM triethanol amine buffer pH 7.6).Conversion is shown in FIG. 1.

After yielding approx. 95 mM dR5P the enzymes were deactivated byheating to 65° C. for 10 min. and excess of AcAld was removed byevaporation. In the second step dR5P in a final concentration of 64 mMwas converted to deoxyadenosine (dA) by adding adenine (A, finalconcentration 58 mM) in the presence of 300 μM MnCl₂, 5 μMGlucose-1.6-bisphosphate, pentosephosphate mutase from E. coli (PPM, 2kU/l), purine nucleoside phosphorylase from E. coli (PNP, 1 kU/l). Thesynthesis was run at 20° C., pH 7.4. In one experiment 200 mM sucroseand 0.6 kU/l sucrose phosphorylase (SP) from Leuconostoc mes. were addedat t=2 h (see arrow in FIG. 2, rhombus, solid line), in a secondexperiment addition of SP was omitted (squares, dotted line). Theconversion was monitored by RP-HPLC (column Hypersil ODS 5 μm, 250×4.6mm; eluent: 30 mM potassium phosphate, 5 mM tetrabutylammoniumhydrogensulfate pH 6.0/1% acetonitrile, flow rate: 1 ml/min,column temp.: 35° C., det.: UV at 260 nm) and is shown in FIG. 2.

EXAMPLE 13

dR5P was prepared by adding FDP-Aldolase II (AldII) from E. coli,pentosephosphate aldolase (PPA) from E. coli and trisosephosphateisomerase (TIM) from E. coli to fructose-1,6-bisphosphate (FDP) andacetaldehyde (AcAld) essentially according to Ex. 6. Excess of AcAld wasremoved by evaporation. dR5P in a final concentration of 60 mM wasconverted to deoxyadenosine (dA) by adding adenine (A, finalconcentration 58 mM) in the presence of 300 μM MnCl₂, 5 μMGlucose-1.6-bisphosphate, pentosephosphate mutase from E. coli (PPM, 1.5kU/l), purine nucleoside phosphorylase from E. coli (PNP, 1 kU/l). Thesynthesis was run at 20° C., pH 7.4. After 24 h sucrose in a finalconcentration of 200 mM and sucrose phosphorylase from Leuconsotoc mes.(1 kU/l) were added. Conversion was monitored by RP-HPLC (dA, A, see ex.12) resp. enzymatical assay (dR5P, using step by stepglycerol-3-phosphate dehydrogenase (GDH), rabbit muscle aldolase (RAMA),trisosephosphate isomerase (TIM), pentosephosphate aldolase (PPA) in thepresence of NADH (0.26 mM in 300 mM Triethanol amine buffer pH 7.6) andphosphomolybdate complexing of inorg. phosphate (Sigma, Proc. No.360-UV). This is shown in FIG. 3.

EXAMPLE 14

dR5P was essentially prepared according to Ex. 6. dR5P in a finalconcentration of 80 mM was then converted to deoxy-6-aminoguanosine(dG-NH₂) by adding 2,6-Diaminopurine (DAP, final concentration 77 mM) inthe presence of 200 mM sucrose, 300 μM MnCl₂, 5 μMGlucose-1.6-bisphosphate, pentosephosphate mutase from E. coli (PPM, 2.5kU/l), purine nucleoside phosphorylase from E. coli (PNP, 1 kU/l),sucrose phosphorylase from Leucoonostoc mes. (SP, 1.5 kU/l). Thesynthesis was run at 20° C. pH 7.4. After 2.5 h, 5 h and 20.5 hadditional amounts of enzymes were added: 2.5 h PPM (2.5 kU/l), PNP (1kU/l, SP (1.5 kU/l), 5 h PPM (2.5 kU/l), SP (1.5 kU/l), 20.5 h: PPM (2.5kU/l), SP (1.5 kU/l). The conversion was monitored by RP-HPLC (columnHypersil ODS 5 μm, 250×4.6 mm; eluent: 30 mM potassium phosphate, 5 mMtetrabutyl ammoniumhydrogensulfate pH 6.0/1% acetonitrile, flow rate: 1ml/min, column temp.: 35° C., det.: UV at 216 nm) and is shown in FIG.4.

1-45. (canceled)
 46. A method for in vitro enzymatic synthesis of adeoxyribonucleoside comprising reacting deoxyribose 1-phosphate (dR1P)and a nucleobase to form a deoxyribonucleoside and an inorganicphosphate.
 47. The method of claim 46, further comprising removing theinorganic phosphate.
 48. The method of claim 46, wherein said reactingcomprises catalyzing said dR1P and said nucleobase with a thymidinephosphorylase (TP, EC 2.4.2.4.) or a purine nucleoside phosphorylase(PNP, EC 2.4.2.1.).
 49. The method of claim 47, comprising removing theinorganic phosphate by one of: (i) converting the inorganic phosphate toinorganic pyrophosphate, (ii) precipitating the inorganic phosphate,(iii) complexing the inorganic phosphate, or (iv) phosphorylating asubstrate with the inorganic phosphate.
 50. The method of claim 47,comprising removing the inorganic phosphate by phosphorylating asubstrate with the inorganic phosphate.
 51. The method of claim 46,wherein the nucleobase is selected from the group consisting of thymine,uracil, adenine, guanine, hypoxanthinine and an analog thereof.
 52. Themethod of claim 51, wherein said analog is selected from the groupconsisting of: 2-thio-uracil, 6-aza-uracil, 5-carboxy-2-thio-uracil,6-aza-thymine, 6-aza-2-thio-thymine and 2,6-diamino-purine.
 53. Themethod of claim 46, further comprising reacting said inorganic phosphatewith fructose-diphosphate (FDP) to form pyrophosphate andfructose-6-phosphate (F6P).
 54. The method of claim 53, wherein saidreacting comprises catalyzing said inorganic phosphate with said FDPwith a Ppi-dependent phosphofructokinase (PFK-Ppi, EC 2.7.1.90).
 55. Themethod of claim 46, further comprising reacting said inorganic phosphatewith a disaccharide to form a monosaccharide and a phosphorylatedmonosaccharide.
 56. The method of claim 55, wherein the disaccharide issucrose or maltose.
 57. The method of claim 56, wherein said reactingcomprises catalyzing said inorganic phosphate and said disaccharide witha sucrose phosphorylase (EC 2.4.1.7) or a maltose phosphorylase (EC2.4.1.8).
 58. The method of claim 46, further comprising generating dR1Pby isomerizing deoxyribose 5-phosphate (dR5P) prior to reacting saiddR1P with a nucleobase.
 59. The method of claim 58, comprisingisomerizing said dR5P with a phosphopentose mutase (PPM, EC 5.4.2.7).60. The method of claim 58, further comprising forming the dR5P bycondensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde prior toisomerizing said dR5P.
 61. The method of claim 60, wherein saidcondensing comprises catalyzing condensing of GAP with acetaldehyde witha phosphopentose aldolase (PPA, EC 4.1.2.4).
 62. The method of claim 60,further comprising enzymatically generating said GAP from fructose1,6-diphosphate, dihydroxyacetone (DHA) or glycerolphosphate (GP) priorto said condensing.
 63. The method of claim 58, comprising generatingsaid deoxyribose 5-phosphate by phosphorylating deoxyribose prior toisomerizing said dR5P.
 64. The method of claim 63, wherein saidphosphorylating comprises catalyzing of deoxyribose with adeoxyribokinase (dRK, EC 2.7.1.15.).
 65. The method of claim 64, whereinsaid dRK is encoded by (a) the nucleotide sequence of SEQ ID NO: 11, (b)a nucleotide sequence encoding the protein encoded by SEQ ID NO: 11 or(c) a nucleotide sequence which hybridizes under stringent conditions tothe complementary sequence of (a) or (b).
 66. The method of claim 62,comprising reacting fructose 1,6-diphosphate with an FDP-aldolase I oran FDP-aldolase II to form said GAP.
 67. The method of claim 62,comprising forming said GAP by reacting DHA with ATP to formdihydroxyacetone phosphate (DHAP), followed by catalyzing isomerizationof said DHAP to GAP with a glycerokinase (GK, EC 2.7.1.30) and a triosephosphate isomerase (TIM, EC 5.3.1.1).
 68. The method of claim 62,comprising forming said GAP by reacting GP with O₂ to formdihydroxyacetone phosphate (DHAP) and H₂O₂, followed by catalyzingisomerization of said DHAP to GAP with a glycerophosphate oxidase (GPO,EC 1.1.3.21) and a triose phosphate isomerase (TIM, EC 5.3.1.1).
 69. Themethod of claim 46, further comprising reacting said deoxyribonucleosidewith a second nucleobase to form a second deoxyribonucleoside containingthe second nucleobase.
 70. The method of claim 69, comprising catalyzingsaid reacting with a nucleoside 2-deoxyribosyl transferase (NdT, EC2.4.2.6).
 71. The method of claim 70, wherein said NdT is encoded by (a)a nucleic acid molecule consisting of the nucleotide sequence of SEQ IDNO: 13, (b) a nucleic acid molecule consisting of a nucleotide sequenceencoding the protein encoded by SEQ ID NO: 13 or (c) a nucleic acidmolecule which hybridizes under stringent conditions to the nucleic acidmolecule of (a) or (b).
 72. The method of claim 69, wherein said secondnucleobase is selected from the group consisting of cytosine and acytosine analog.
 73. The method of claim 69, wherein said secondnucleobase is selected from the group consisting of 5-aza-cytosine,2,6-dichloro-purine, 6-aza-thymine and 5-fluoro-uracil.
 74. A method forthe in vitro enzymatic synthesis of a deoxyribonucleoside comprising:(i) condensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde toform deoxyribose 5-phosphate (dR5P), (ii) isomerizing said dR5P todeoxyribose 1-phosphate (dR1P), and (iii) reacting said dR1P and anucleobase, to form said deoxyribonucleoside and an inorganic phosphate.75. The method of claim 74, further comprising removing the inorganicphosphate.
 76. The method of claim 75, comprising removing the inorganicphosphate by phosphorylation of a substrate with the inorganicphosphate.
 77. The method of claim 74, comprising carrying out thecomplete reaction of steps (i) to (iii) without isolating intermediateproducts.
 78. The method of claim 74, comprising generating said GAPfrom fructose 1,6-diphosphate (FDP), dihydroxy-acetone (DHA) orglycerolphosphate (GP) prior to said condensing of GAP.
 79. The methodof claim 74, further comprising removing excess acetaldehyde before step(ii).
 80. The method of claim 78, further comprising generating said GAPand removing excess starting materials or by-products before step (ii).81. The method of claim 80, wherein said excess starting material isfructose 1,6-diphosphate and said excess by-product is deoxyxylulose1-phosphate (dX1P).
 82. The method of claim 78, comprising generatingGAP from FDP, and generating DXP1 as an excess by-product thereby.
 83. Amethod for the in vitro enzymatic synthesis of a deoxyribonucleosidecomprising: (i) phosphorylating deoxyribose to deoxyribose 5-phosphate(dR5P), (ii) isomerizing said dR5P to deoxyribose 1-phosphate (dR1P),and (iii) reacting said dR1P and a nucleobase to form saiddeoxyribonucleoside and an inorganic phosphate.
 84. The method of claim83, further comprising removing the inorganic phosphate.
 85. The methodof claim 84, comprising removing the inorganic phosphate byphosphorylating a substrate with the inorganic phosphate.
 86. The methodof claim 83, comprising conducting the complete reaction of steps (i) to(iii) without isolating intermediate products.
 87. A method forpreparing an enzyme for an in vitro method for enzymatic synthesis of adeoxyribonucleoside, comprising reacting (i) an isolated nucleic acidmolecule encoding a nucleoside 2-deoxyribosyl transferase (NdT, EC2.4.2.6) with (ii) a deoxyribonucleoside containing a first nucleobase,wherein said nucleic acid molecule comprises (a) the nucleotide sequenceshown in SEQ ID NO: 13, (b) a nucleotide sequence encoding the proteinencoded by SEQ ID NO: 13 or (c) a nucleotide sequence hybridizing understringent conditions to the complementary sequence of (a) or (b), andwherein said deoxyribonucleoside containing a first nucleobase isfurther reacted with a second nucleobase to form a deoxyribonucleosidecontaining said second nucleobase.
 88. The method of claim 87, whereinthe second nucleobase is selected from the group consisting of cytidineand a cytidine analog.
 89. The method of claim 88, wherein the analog isselected from the group consisting of: 6-methyl purine,2-amino-6-methylmercaptopurine, 6-dimethylaminopurine, 5-azacytidine,2,6-dichloropurine, 6-chloroguanine, 6-chloropurine, 6-azathymine,5-fluorouracil, ethyl-4-amino-5-imidazole carboxylate,imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide.
 90. The methodof claim 87, wherein the first nucleobase is selected from the groupconsisting of adenine, guanine, thymine, uracil and hypoxanthine. 91.The method of claim 87, comprising containing the nucleic acid moleculein a recombinant vector in operative linkage with an expression controlsequence.
 92. The method of claim 81, comprising containing the nucleicacid in a recombinant cell.
 93. A method for preparing an enzyme for anin vitro method for enzymatic synthesis of a deoxyribonucleoside,comprising reacting (i) an isolated nucleic acid molecule encoding adeoxyribokinase (dRK, EC 2.7.1.5) with (ii) deoxyribose, furthercomprising phosphorylating said deoxyribose to deoxyribose 5-phosphate,wherein said nucleic acid molecule comprises (a) the nucleotide sequenceshown in SEQ ID NO: 11, (b) a nucleotide sequence encoding the proteinencoded by SEQ ID NO: 11 or (c) a nucleotide sequence hybridizing understringent conditions to the complementary sequence of (a) or (b).
 94. Amethod for synthesizing a deoxyribonucleoside in vitro, comprisingcontacting a mixture containing deoxyribose and phosphate with an enzymehaving NdT activity to form deoxyribose 5-phosphate and obtainingdeoxyribose 5-phosphate therefrom.